Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR)

MicroRNAs (miRNAs) are small (～22 nt) RNAs that play important roles in gene regulatory networks by binding to and repressing the activity of specific target mRNAs. Recent studies have indicated that miRNAs circulate in a stable, cell-free form in the bloodstream and that the abundance of specific miRNAs in plasma or serum can serve as biomarkers of cancer and other diseases. Measurement of circulating miRNAs as biomarkers is associated with some special challenges, including those related to pre-analytic variation and data normalization. We describe here our procedure for qRT-PCR analysis of circulating miRNAs as biomarkers, and discuss relevant issues of sample preparation, experimental design and data analysis.     

NF-kappaB in breast cancer cells promotes osteolytic bone metastasis by inducing osteoclastogenesis via GM-CSF

Advanced breast cancers frequently metastasize to bone, resulting in osteolytic lesions, yet the underlying mechanisms are poorly understood. Here we report that nuclear factor-kappaB (NF-kappaB) plays a crucial role in the osteolytic bone metastasis of breast cancer by stimulating osteoclastogenesis. Using an in vivo bone metastasis model, we found that constitutive NF-kappaB activity in breast cancer cells is crucial for the bone resorption characteristic of osteolytic bone metastasis. We identified the gene encoding granulocyte macrophage-colony stimulating factor (GM-CSF) as a key target of NF-kappaB and found that it mediates osteolytic bone metastasis of breast cancer by stimulating osteoclast development. Moreover, we observed that the expression of GM-CSF correlated with NF-kappaB activation in bone-metastatic tumor tissues from individuals with breast cancer. These results uncover a new and specific role of NF-kappaB in osteolytic bone metastasis through GM-CSF induction, suggesting that NF-kappaB is a potential target for the treatment of breast cancer and the prevention of skeletal metastasis.     

Allosteric modulation of kinases and GPCRs: design principles and structural diversity

Allosteric binding sites, as opposed to traditional orthosteric binding sites, offer unparalleled opportunities for drug discovery by providing high levels of selectivity, mimicking physiological conditions, affording fewer side effects because of desensitization/downregulation, and engendering ligands with chemotypes divergent from orthosteric ligands. For kinases, allosteric mechanisms described to date include alteration of protein kinase conformation blocking productive ATP binding which appear 'ATP competitive' or blocking kinase activation by conformational changes that are 'ATP non-competitive'. For GPCRs, allosteric mechanisms impart multiple modes of target modulation (positive allosteric modulation (PAM), negative allosteric modulation (NAM), neutral cooperativity, partial antagonism (PA), allosteric agonism and allosteric antagonism). Here, we review recent developments in the design principles and structural diversity of allosteric ligands for kinases and GPCRs.     

TREEasy: An automated workflow to infer gene trees,species trees,and phylogenetic networks from multilocus data

Multilocus genomic data sets can be used to infer a rich set of information about the evolutionary history of a lineage, including gene trees, species trees, and phylogenetic networks. However, user‐friendly tools to run such integrated analyses are lacking, and workflows often require tedious reformatting and handling time to shepherd data through a series of individual programs. Here, we present a tool written in Python—TREEasy—that performs automated sequence alignment (with MAFFT), gene tree inference (with IQ‐Tree), species inference from concatenated data (with IQ‐Tree and RaxML‐NG), species tree inference from gene trees (with ASTRAL, MP‐EST, and STELLS2), and phylogenetic network inference (with SNaQ and PhyloNet). The tool only requires FASTA files and nine parameters as inputs. The tool can be run as command line or through a Graphical User Interface (GUI). As examples, we reproduced a recent analysis of staghorn coral evolution, and performed a new analysis on the evolution of the “WGD clade” of yeast. The latter revealed novel patterns that were not identified by previous analyses. TREEasy represents a reliable and simple tool to accelerate research in systematic biology ( https://github.com/MaoYafei/TREEasy ).     

Primate Location Preference in a Double-Tier Cage: The Effects of Illumination and Cage Height

Nonhuman primates are frequently housed in double-tier arrangements with significant differences between the environments of the upper and lower-row cages. Although several studies have investigated whether this arrangement alters monkeys' behavior, no studies have addressed the two most notable differences, light and height, individually to determine their relative importance. This experiment examined how rhesus and long-tailed macaques allocated their time between the upper and lower-row cages of a 1-over-1 apartment module under different lighting conditions. In Condition A, monkeys' baseline degree of preference for the upper- and lower-row was tested. In Condition B, the lighting environment was reversed by limiting illumination in the upper-row cage and increasing illumination in the lower-row cage. In both conditions, monkeys spent more time in the upper-row cage, thus indicating a strong preference for elevation regardless of illumination. The amount of time that monkeys spent in the lower-row cage increased by 7% under reversed lighting, but this trend was not significant. These results corroborate the importance of providing captive primates with access to elevated areas.     

Extreme climatic events constrain space use and survival of a ground‐nesting bird

Two fundamental issues in ecology are understanding what influences the distribution and abundance of organisms through space and time. While it is well established that broad‐scale patterns of abiotic and biotic conditions affect organisms’ distributions and population fluctuations, discrete events may be important drivers of space use, survival, and persistence. These discrete extreme climatic events can constrain populations and space use at fine scales beyond that which is typically measured in ecological studies. Recently, a growing body of literature has identified thermal stress as a potential mechanism in determining space use and survival. We sought to determine how ambient temperature at fine temporal scales affected survival and space use for a ground‐nesting quail species (Colinus virginianus; northern bobwhite). We modeled space use across an ambient temperature gradient (ranging from ?20 to 38 °C) through a maxent algorithm. We also used Andersen–Gill proportional hazard models to assess the influence of ambient temperature‐related variables on survival through time. Estimated available useable space ranged from 18.6% to 57.1% of the landscape depending on ambient temperature. The lowest and highest ambient temperature categories (35 °C, respectively) were associated with the least amount of estimated useable space (18.6% and 24.6%, respectively). Range overlap analysis indicated dissimilarity in areas where Colinus virginianus were restricted during times of thermal extremes (range overlap = 0.38). This suggests that habitat under a given condition is not necessarily a habitat under alternative conditions. Further, we found survival was most influenced by weekly minimum ambient temperatures. Our results demonstrate that ecological constraints can occur along a thermal gradient and that understanding the effects of these discrete events and how they change over time may be more important to conservation of organisms than are average and broad‐scale conditions as typically measured in ecological studies.     

A decade of boreal rich fen greenhouse gas fluxes in response to natural and experimental water table variability

Rich fens are common boreal ecosystems with distinct hydrology, biogeochemistry and ecology that influence their carbon (C) balance. We present growing season soil chamber methane emission (F_CH4), ecosystem respiration (ER), net ecosystem exchange (NEE) and gross primary production (GPP) fluxes from a 9‐years water table manipulation experiment in an Alaskan rich fen. The study included major flood and drought years, where wetting and drying treatments further modified the severity of droughts. Results support previous findings from peatlands that drought causes reduced magnitude of growing season F_CH4, GPP and NEE, thus reducing or reversing their C sink function. Experimentally exacerbated droughts further reduced the capacity for the fen to act as a C sink by causing shifts in vegetation and thus reducing magnitude of maximum growing season GPP in subsequent flood years by ~15% compared to control plots. Conversely, water table position had only a weak influence on ER, but dominant contribution to ER switched from autotrophic respiration in wet years to heterotrophic in dry years. Droughts did not cause inter‐annual lag effects on ER in this rich fen, as has been observed in several nutrient‐poor peatlands. While ER was dependent on soil temperatures at 2 cm depth, F_CH4 was linked to soil temperatures at 25 cm. Inter‐annual variability of deep soil temperatures was in turn dependent on wetness rather than air temperature, and higher F_CH4 in flooded years was thus equally due to increased methane production at depth and decreased methane oxidation near the surface. Short‐term fluctuations in wetness caused significant lag effects on F_CH4, but droughts caused no inter‐annual lag effects on F_CH4. Our results show that frequency and severity of droughts and floods can have characteristic effects on the exchange of greenhouse gases, and emphasize the need to project future hydrological regimes in rich fens.